Flattened-Top Domical Water Drops Formed through Self-Organization of Hydrophobin Membranes: A Structural and Mechanistic Study Using Atomic Force Microscopy Ryota Yamasaki,†,‡,§ Yoshiyuki Takatsuji,†,‡,§ Hitoshi Asakawa,§,∥,⊥ Takeshi Fukuma,§,∥,⊥ and Tetsuya Haruyama*,†,‡,§ †
Division of Functional Interface Engineering, Department of Biological Functions Engineering, and ‡Research Center for Eco-fitting Technology, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Fukuoka 808-0196, Japan § Advanced Catalytic Transformation Program for Carbon Utilization (ACT-C), Japan Science and Technology Agency (JST), Tokyo 102-0076, Japan ∥ Division of Electrical Engineering and Computer Science and ⊥Bio-AFM Frontier Research Center, Kanazawa University, Kanazawa 920-1192, Japan S Supporting Information *
ABSTRACT: The Trichoderma reesei hydrophobin, HFBI, is a unique structural protein. This protein forms membranes by self-organization at air/ water or water/solid interfaces. When HFBI forms a membrane at an air/water interface, the top of the water droplet is flattened. The mechanism underlying this phenomenon has not been explored. In this study, this unique phenomenon has been investigated. Self-organized HFBI membranes form a hexagonal structured membrane on the surface of water droplets; the structure was confirmed by atomic force microscopy (AFM) measurement. Assembled hexagons can form a planar sheet or a tube. Self-organized HFBI membranes on water droplets form a sheet with an array of hexagonal structures or a honeycomb structure. This membrane, with its arrayed hexagonal structures, has very high buckling strength. We hypothesized that the high buckling strength is the reason that water droplets containing HFBI form flattened domes. To test this hypothesis, the strength of the self-organized HFBI membranes was analyzed using AFM. The buckling strength of HFBI membranes was measured to be 66.9 mN/m. In contrast, the surface tension of water droplets containing dissolved HFBI is 42 mN/m. Thus, the buckling strength of a selforganized HFBI membrane is higher than the surface tension of water containing dissolved HFBI. This mechanistic study clarifies why the water droplets formed by self-organized HFBI membranes have a flattened top. KEYWORDS: self-organized membrane, HFBI, surface tension, surface activity, atomic force microscopy, force curve measurement
S
produced in the cell walls of fungi and is a small amphiphilic molecule.4−6 In particular, HFBI, a species of hydrophobin protein, can form very stable self-organized monolayers at air/ water interfaces. In our previous studies, we showed that HFBI forms two-dimensional ordered structures through molecular self-organization at air/water interfaces, and the liquid droplets formed show a particular shape. Szilvay et al. investigated HFBI self-organization on air/water interfaces using a hanging drop method and atomic force microscopy (AFM) imaging.7 Kisko
elf-organization is a remarkable property that allows molecules to form intricate structures. Many proteins efficiently undergo such structural organization, and some form extraordinarily accurate structures through selforganization. Tobacco mosaic virus capsid protein, for example, is a well-known self-organizing protein that forms a highly precise structure.1 Ferritin is also known to be a self-organizing protein that stores Fe in living systems.2,3 These self-organizing proteins form sterically fixed structures in homogeneous solutions and have been well-studied in the past. In contrast to self-organization in homogeneous systems, we have focused on a fungus-derived self-organizing protein, hydrophobin, which is self-organizing at phase interfaces, such as air/water or water/solid interfaces. The interface protein hydrophobin is © 2015 American Chemical Society
Received: July 2, 2015 Accepted: November 23, 2015 Published: November 23, 2015 81
DOI: 10.1021/acsnano.5b04049 ACS Nano 2016, 10, 81−87
Article
www.acsnano.org
Article
ACS Nano
Figure 1. (a) HFBI protein. Hydrophobic residues are colored gray. (b) Photographs of the domical shape of an HFBI-containing water droplet (left) and a flattened droplet in which an HFBI self-organized structure has developed at the surface (right). (c) Hypothetical illustration of HFBI self-organization and the process of water droplet flattening.
et al. investigated the structure of a self-organized HFBI membrane at the air/water interface using grazing-incidence Xray diffraction (GID).8 Self-organized HFBI monolayer membranes can be transferred onto a solid surface by stamping the droplet onto the solid using either an upward process7 or a downward process.9 Moreover, HFBI can be employed as a molecular carrier to form nanoaccurate structures. If the charges of the amino acids in HFBI are changed or it is tagged with functional proteins using a genetic engineering technique, the HFBI domain acts as a molecular carrier to form a stable self-organized membrane, in a manner similar to that of the HFBI molecule alone, as reported in our previous study.10−12 In this study, we focused on the unique flattened shape of the water droplets formed by HFBI self-organization. Typically, water droplets form a spherical shape due to surface tension. Water droplets on a solid substrate form a domical shape. In contrast, a droplet including HFBI on a solid substrate forms a domical shape with a flattened top. Flattened water droplets are a unique phenomenon, and the underlying mechanisms are yet to be explored. Here, we investigated this unique phenomenon. The structure of self-organized HFBI membranes was measured using AFM, and mechanical analysis of this membrane was also performed using force curve measurements by AFM.
Figure 2. Photographs of HFBI-containing water droplets during the process of HFBI self-organization over time. Top: Bright-field edge-on image. Middle: Bright-field image from above. Bottom: Fluorescent image from above.
RESULTS AND DISCUSSION Water Droplet Development of an HFBI Self-Organized Membrane at the Air/Water Interface. As can be observed in Figure 1b, water droplets containing dissolved HFBI form a domical shape with a flattened top; water without HFBI forms a domical shape. Thus, the area of the flattened top expands with time (Figure 2). Figure 1c illustrates the process of HFBI self-organization. Three types of images, a bright-field image from edge-on, an image from directly above, and a fluorescent image from directly above, show the flattened-top domical drops formed by the HFBI solution (Figure 2). It is clear from the images that the flat surface is at the top of the domical drop. In the case of fluorescent images, fluorescein isothiocyanate (FITC)-stained HFBI protein was used and irradiated with light at a wavelength of 535 nm. An organized membrane on the flat top of the domical drops can be observed. The observations strongly suggest that the HFBI molecules are localized and self-organized at the top of the domical drop (at the air/water interface). The HFBI molecule rafts continuously moved toward the center of the droplets because of convection driven by differential evaporation. The
flat shape is probably dependent on the high structural-buckling strength of the HFBI self-organized membrane. The selforganized HFBI membrane has a precise structure, which may provide a high buckling strength at the air/water interface. If the buckling strength of the self-organized HFBI membrane was lower than the surface tension of the domical drop, the domical drop would not be flat, even though the top was covered by the membrane. Surface Tension and Self-Organization Ratio of HFBIContaining Water Droplets. As shown in the side-view image of an HFBI droplet (Figure 1), the flat part of the domical drop is horizontal. The shape of the domical drop is determined by the surface tension and the interfacial tension between the droplet solution and the solid substrate, and it is likely influenced by gravity, as well. The right axis of Figure 3 (shown as squares) shows the shape changes of a domical drop over time. The self-organization ratio is defined as “(a) length of upper base” divided by “(b) length of lower base”. The left axis (shown as spheres) shows the surface tension of the 82
DOI: 10.1021/acsnano.5b04049 ACS Nano 2016, 10, 81−87
Article
ACS Nano
surfactant-containing droplet spread on the FTO substrate because the surfactant activated the surface of the FTO. However, although HFBI is expected to behave as a surfactant, HFBI droplets did not spread but retained their shape. One of the possible explanations, as can be seen Figure 5a, is that three vectors are in play, and these forces equilibrate at the three phase interfaces of the droplet.14,15 γSV = γLS + γLV cos θ
If a droplet contains a surfactant, it will expand on a solid substrate. As γLS decreases, the droplet spreads toward γSV (Figure 5b). In the case of HFBI, the molecules are large and globular in shape compared with the surfactant. Therefore, HFBI cannot enter the interspace; the forces at the three phase interfaces remain in equilibrium, and the liquid does not spread (Figure 5c). The other protein (glucose oxidase, GOx) also did not spread on FTO (Figure S2). These results suggest that the size and shape of the interface activation molecules are important in activating the surface, allowing the liquid to spread. Structural Analysis of a Self-Organized HFBI Membrane. Self-organized HFBI submerged in 100 mM phosphate buffer at pH 7.0 was also visualized using an atomic force microscope. The self-organized membrane was transferred to a highly oriented pyrolytic graphite (HOPG) substrate from the top of the domical droplet (flat portion), as shown in Figure 6c. The HFBI self-organized membrane was observed to have a perfect honeycomb structure in the submerged AFM image. This result indicates that the domical droplet was flattened by the rigid structure of the HFBI self-organized membrane. A hexagonal structure can form either a planar surface or a tube but not a sphere (Supporting Information). Elastic Buckling Strength Measurement of a SelfOrganized HFBI Membrane. The AFM measurement suggested that the self-organized HFBI membrane is very strong (Figure 6c). In this study, we tried to quantify the buckling strength of a self-organized HFBI membrane. To investigate this issue, a Nuclepore membrane was employed as a substrate to perform a force curve measurement using AFM. The Nuclepore membrane contains many perforations (average diameter = 0.2 μm). To measure buckling strength, a selforganized HFBI membrane was placed on the Nuclepore membrane and then was observed by AFM (Figure 7). As shown in the AFM images, two of the three visible holes were covered with a self-organized HFBI membrane. A height profile is shown under the AFM image. From this profile, it was clear that one of the holes was covered with the HFBI membrane. Next, a hole covered with an HFBI membrane was pressed down with an AFM tip, and the force curve was measured (Figure 8a). The force curve measurement indicated a membrane rupture. After the force curve measurement, the same point was imaged (Figure 7), and it was confirmed that the HFBI membrane covering the hole was broken by the AFM tip. These measurements were conducted using a qp-BioAC probe (spring constant = 0.1 N/m). Another hole covered with an HFBI membrane was also measured (Figure S3), and another position of the self-organized HFBI membrane on the Nuclepore membrane hole was also measured (Figure S4). From the force curve measurements, the force required to break the HFBI membrane was 1.34 nN. This value was divided by the diameter of the contact area diameter (the radius of curvature of an AFM tip is
